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  • 93
    Jena Bioscience mant gtp
    Mant Gtp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mant gtp/product/Jena Bioscience
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mant gtp - by Bioz Stars, 2021-03
    93/100 stars
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    N/A
    This Ambion ribonucleotide triphosphate rNTP solution is a component of the MAXIscript Kits SKU s AM1312 AM1316 AM1314 AM1326 AM1324 AM1318 AM1322 AM1320 AM1310 and AM1308 and is supplied in
      Buy from Supplier

    93
    Millipore gtp
    Gtp, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gtp/product/Millipore
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gtp - by Bioz Stars, 2021-03
    93/100 stars
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    86
    PerkinElmer α 32 p gtp
    Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, <t>GTP,</t> or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.
    α 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p gtp/product/PerkinElmer
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α 32 p gtp - by Bioz Stars, 2021-03
    86/100 stars
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    86
    GE Healthcare α 32 p gtp
    Inhibition by tetracycline of the (p)ppGpp synthase activity of pea chloroplast extract. ( A ) The S-30 fractions of pea chloroplasts and E.coli W3110 cells were assayed for (p)ppGpp synthase activity with [α- 32 <t>P]GTP</t> as substrate in the presence of tetracycline at concentrations of 0 μM (lanes 1 and 5), 50 μM (lanes 2 and 6), 250 μM (lanes 3 and 7) or 500 μM (lanes 4 and 8). Labeled nucleotides were separated by 1D-TLC on PEI-cellulose and visualized by autoradiography (12 h exposure). ( B ) Longer exposure (36 h) of the TLC plate shown in (A). ( C and D ) The intensities of the ppGpp spots obtained with the S-30 fraction of E.coli (C) or of the pppGpp spots obtained with the S-30 fraction of pea chloroplasts (D) were measured for the autoradiogram shown in (B). Data are expressed relative to the corresponding values for the incubations performed in the absence of tetracycline, and are representative of experiments performed with three independent chloroplast or bacterial extracts.
    α 32 P Gtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p gtp/product/GE Healthcare
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    α 32 p gtp - by Bioz Stars, 2021-03
    86/100 stars
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    N/A
    Guanosine triphosphate labeled on the alpha phosphate group with 33P 33P has lower energy than 32P and is sometimes used instead of 32P to improve resolution in autoradiography Common applications
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    Image Search Results


    Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Purification, Binding Assay, Thin Layer Chromatography, Labeling

    In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: In Vitro, Competitive Binding Assay, Binding Assay, Purification, Labeling, Thin Layer Chromatography

    HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Binding Assay, Competitive Binding Assay

    Inhibition by tetracycline of the (p)ppGpp synthase activity of pea chloroplast extract. ( A ) The S-30 fractions of pea chloroplasts and E.coli W3110 cells were assayed for (p)ppGpp synthase activity with [α- 32 P]GTP as substrate in the presence of tetracycline at concentrations of 0 μM (lanes 1 and 5), 50 μM (lanes 2 and 6), 250 μM (lanes 3 and 7) or 500 μM (lanes 4 and 8). Labeled nucleotides were separated by 1D-TLC on PEI-cellulose and visualized by autoradiography (12 h exposure). ( B ) Longer exposure (36 h) of the TLC plate shown in (A). ( C and D ) The intensities of the ppGpp spots obtained with the S-30 fraction of E.coli (C) or of the pppGpp spots obtained with the S-30 fraction of pea chloroplasts (D) were measured for the autoradiogram shown in (B). Data are expressed relative to the corresponding values for the incubations performed in the absence of tetracycline, and are representative of experiments performed with three independent chloroplast or bacterial extracts.

    Journal: Nucleic Acids Research

    Article Title: Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline

    doi: 10.1093/nar/gkh916

    Figure Lengend Snippet: Inhibition by tetracycline of the (p)ppGpp synthase activity of pea chloroplast extract. ( A ) The S-30 fractions of pea chloroplasts and E.coli W3110 cells were assayed for (p)ppGpp synthase activity with [α- 32 P]GTP as substrate in the presence of tetracycline at concentrations of 0 μM (lanes 1 and 5), 50 μM (lanes 2 and 6), 250 μM (lanes 3 and 7) or 500 μM (lanes 4 and 8). Labeled nucleotides were separated by 1D-TLC on PEI-cellulose and visualized by autoradiography (12 h exposure). ( B ) Longer exposure (36 h) of the TLC plate shown in (A). ( C and D ) The intensities of the ppGpp spots obtained with the S-30 fraction of E.coli (C) or of the pppGpp spots obtained with the S-30 fraction of pea chloroplasts (D) were measured for the autoradiogram shown in (B). Data are expressed relative to the corresponding values for the incubations performed in the absence of tetracycline, and are representative of experiments performed with three independent chloroplast or bacterial extracts.

    Article Snippet: Either [α-32 P]GTP, [γ-32 P]ATP or [γ-32 P]GTP (each with a specific activity of 3000 Ci/mmol; Amersham Biosciences) at a concentration of 10 μCi/ml was used as a donor for radioisotope labeling.

    Techniques: Inhibition, Activity Assay, Labeling, Thin Layer Chromatography, Autoradiography

    Association of (p)ppGpp synthase activity with 70S ribosomes in pea chloroplasts. ( A ) Schematic representation of the procedure for fractionation of pea chloroplast and bacterial cell extracts. ( B ) Assay of the chloroplast and E.coli W3110 P-150(suc) fractions for (p)ppGpp synthase activity with [α- 32 P]GTP as substrate. The chloroplast P-150, S-150, P-150(wash), and S-150(wash) fractions, and the reconstituted fraction consisting of chloroplast P-150(wash) and S-150(wash) were examined. The reaction mixtures were subjected to 2D-TLC and autoradiography. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. Relative intensities of the pppGpp spots appeared from the chloroplast fractions were: P-150, 100; S-150, 2.6; P-150(suc), 4.6; P-150(wash), 17.2; S-150(wash), 5.8; P-150(wash) + S-150(wash), 49.9. Spot intensities were quantitated based on the densitometric analysis as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline

    doi: 10.1093/nar/gkh916

    Figure Lengend Snippet: Association of (p)ppGpp synthase activity with 70S ribosomes in pea chloroplasts. ( A ) Schematic representation of the procedure for fractionation of pea chloroplast and bacterial cell extracts. ( B ) Assay of the chloroplast and E.coli W3110 P-150(suc) fractions for (p)ppGpp synthase activity with [α- 32 P]GTP as substrate. The chloroplast P-150, S-150, P-150(wash), and S-150(wash) fractions, and the reconstituted fraction consisting of chloroplast P-150(wash) and S-150(wash) were examined. The reaction mixtures were subjected to 2D-TLC and autoradiography. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. Relative intensities of the pppGpp spots appeared from the chloroplast fractions were: P-150, 100; S-150, 2.6; P-150(suc), 4.6; P-150(wash), 17.2; S-150(wash), 5.8; P-150(wash) + S-150(wash), 49.9. Spot intensities were quantitated based on the densitometric analysis as described in Materials and Methods.

    Article Snippet: Either [α-32 P]GTP, [γ-32 P]ATP or [γ-32 P]GTP (each with a specific activity of 3000 Ci/mmol; Amersham Biosciences) at a concentration of 10 μCi/ml was used as a donor for radioisotope labeling.

    Techniques: Activity Assay, Fractionation, Thin Layer Chromatography, Autoradiography

    Assay of the S-30 fractions of pea chloroplasts and E.coli cells for (p)ppGpp synthase activity. The S-30 fractions of pea chloroplasts ( A , E , I ) or of E.coli strains W3110 ( B , F , J ) or CF1678 ( C , G , K ) were incubated with [α- 32 P]GTP (A, B, C), [γ- 32 P]ATP (E, F, K) or [γ- 32 P]GTP (I, J, K), after which 32 P-labeled nucleotides were separated by 2D-TLC on PEI-cellulose sheets and visualized by autoradiography. The origin and the positions of spots corresponding to ADP, ATP, GMP, GDP, GTP, ppGpp, pppGpp and orthophosphate are indicated. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. As a negative control, the 2D-TLC profile of each nucleotide used for labeling is shown on the right end of each panel as ‘no extract’ ( D , H , L ).

    Journal: Nucleic Acids Research

    Article Title: Guanosine tetra- and pentaphosphate synthase activity in chloroplasts of a higher plant: association with 70S ribosomes and inhibition by tetracycline

    doi: 10.1093/nar/gkh916

    Figure Lengend Snippet: Assay of the S-30 fractions of pea chloroplasts and E.coli cells for (p)ppGpp synthase activity. The S-30 fractions of pea chloroplasts ( A , E , I ) or of E.coli strains W3110 ( B , F , J ) or CF1678 ( C , G , K ) were incubated with [α- 32 P]GTP (A, B, C), [γ- 32 P]ATP (E, F, K) or [γ- 32 P]GTP (I, J, K), after which 32 P-labeled nucleotides were separated by 2D-TLC on PEI-cellulose sheets and visualized by autoradiography. The origin and the positions of spots corresponding to ADP, ATP, GMP, GDP, GTP, ppGpp, pppGpp and orthophosphate are indicated. Data are representative of experiments performed with three independent chloroplast or bacterial extracts. As a negative control, the 2D-TLC profile of each nucleotide used for labeling is shown on the right end of each panel as ‘no extract’ ( D , H , L ).

    Article Snippet: Either [α-32 P]GTP, [γ-32 P]ATP or [γ-32 P]GTP (each with a specific activity of 3000 Ci/mmol; Amersham Biosciences) at a concentration of 10 μCi/ml was used as a donor for radioisotope labeling.

    Techniques: Activity Assay, Incubation, Labeling, Thin Layer Chromatography, Autoradiography, Negative Control